Methods and compositions for delivering therapeutic agents to the central nervous system

ABSTRACT

Methods are provided for targeting therapeutic agents to an injury site within the central nervous system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) to U.S.Provisional Application No. 61/151,116, filed Feb. 9, 2009, which isincorporated herein by reference in its entirety.

This application is related to U.S. patent application Ser. No.10/232,908 titled “METHOD FOR ERADICATING PAIN OF CENTRAL ORIGINRESULTING FROM SPINAL CORD INJURY,” filed Aug. 30, 2002, which is herebyincorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

a. Field of the Invention

The invention generally relates to methods and compositions for therapyin the central nervous system (CNS) and more particularly to methods foridentifying target sites for delivery of these therapies into the CNS ofa patient in need thereof.

b. Background Art

Acute and/or chronic injury to the spinal cord often debilitatespatients, altering the patient's everyday life. Loss of motion due tonerve damage and/or disabling pain often results from spinal cordinjury. Recovered function from damaged or necrotic tissue within thespinal cord has proven to be problematic, often leaving patients withspinal injuries in a state of chronic disability for the remainder oftheir lives.

Over the past several years a number of studies have focused onrestorative and regenerative therapies with regard to spinal cordinjury. These studies have focused on the potential for restoring orregenerating damaged tissue using animal models (See Schwab, Repairingthe Injured Spinal Cord, SCIENCE 295 (5557):1029-1031; Almudena et al.,Functional Recovery of Paraplegic Rats and Motor Axon Regeneration inTheir Spinal Cords by Olfactory Ensheathing Glia, NEURON 25(2), (2000),425-435; Kwon et al., Animal Models Used in Spinal Cord RegenerationResearch, SPINE, 27(14) (2002):1504-1510). However, translation ofrestorative and regenerative therapies in spinal cord injury, derivedfrom animal research, to the human condition, remains a significantchallenge.

A significant difficulty faced in providing adequate restorative andregenerative therapies to the CNS is directed at how to target therapieswithin an acutely or chronically injured site to maximize the effect ofthe therapy, i.e., one must have a method of targeting, during surgeryon, or manipulation of, the CNS, for placement of therapeutic agentswithin the injury site. For example, after spinal cord injury, variousdegrees of cell death, partial injury, progressive injury,demyelization, glial scarring, and hematoma formation may occur. (See,Åkesson, Human Spinal Cord Transplantation, Experimental and ClinicalApplication, Stockholm 2000, Division of Geriatric Medicine, NEUROTEC,pp 7-16). Each of these different cellular environments providesdifferent challenges for useful therapeutic agents, includingrestorative cellular therapies. For example, for a therapeutic agent tobe effective, the cellular content and environment must be optimized formaximal potential benefit of that therapy. In this regard, certaintherapies, e.g., drugs or stem cell compositions, would have littlebenefit if they would not have the capacity or numbers to influence theinjury, or be delivered into a spinal region with a hostile environmentfor cell survival, e.g., a hematoma. In such circumstances the cellswill often undergo apoptosis or become quiescent.

Against this backdrop the present disclosure was developed.

SUMMARY

In various embodiments, methods of delivering a therapeutic agent tospecific locations in the central nervous system (CNS) of a patient areprovided. The methods combine imaging the site of damage or injury,detecting neuroelectrical activity at the injury, and then deliveringtherapeutic agents to the sites. In certain embodiments, a preoperativeimage of the CNS or a portion thereof (e.g. the spinal cord) of apatient is acquired to determine the boundaries of a region of damagedtissue. One or more intraoperative ultrasound images of an injury regionare then obtained. The neuroelectrical activity at and surrounding theinjury site is measured. In certain cases, zones of active neuronactivity, inactivity, hypoactivity and/or hyperactivity are detected.The ultrasound images are correlated with the measured neuroelectricalactivity at and surrounding the injury site to identify one or moredelivery zones for said therapeutic agent.

Various therapeutic agents can promote axonal regeneration orremyelination, inhibit nerve impulses from dissipating in demyelinatedareas, or inhibit exotoxic shock. In still further variations thetherapeutic agents are stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary MRI from a patient having a spinal cordinjury.

FIG. 2 depicts an exemplary intraoperative ultrasound from a patienthaving a spinal cord injury.

FIG. 3A depicts an electrophysiologic recording of an injured spinalcord at a contusion site analysis of the dorsal grey matter (DREZ)within an injury site.

FIG. 3B depicts an electrophysiologic recording of spontaneousneuroelectrical activity for normal or healthy cells in the dorsal greymatter of the spinal cord caudal to an injury site

FIG. 3C depicts a region of cells in the grey matter (DREZ) adjacent to,or bordering, the acute injury showing hyperactive neuroelectricalactivity.

FIG. 4A depicts an electrophysiologic recording of uninjured tissue inthe spinal thalamic tract of an individual.

FIG. 4B depicts an electrophysiologic recording of uninjured tissue inthe lateral posterior column of an individual.

FIG. 4C depicts an electrophysiologic recording of normal, uninjuredtissue in the medial posterior column.

FIG. 5A depicts an electrophysiologic recording of the corticospinaltract of a patient.

FIG. 5B depicts an electrophysiologic recording of the dorsal greymatter of a patient.

FIG. 6 depicts an electrical recording of extreme myelomalacia andcystic necrosis in the tissue of a patient.

FIG. 7 depicts an electrophysiological recording of a probe detecting adorsal grey matter.

FIG. 8 depicts an electrophysiological recording of a probe detecting aa dorsal root entry zone (DREZ).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides compositions and methods for deliveringtherapies and therapeutic agents useful in the treatment of CNS defects,including injuries. As referred to herein, the terms “Central NervousSystem” or “CNS” refer to the spinal cord and brain.

In one embodiment, the disclosure provides a method of identifying oneor more delivery sites for a therapeutic agent in the CNS of a patient.Preoperative imaging data of the CNS of the patient is first obtained todetermine boundaries of a defective region (e.g. injury site) in the CNSof the patient. One or more intraoperative ultrasound images of theregion of injury are then obtained to delineate the boundaries of theinjury site. The neuroelectrical activity in one or more injury tractsis then measured to identify zones of active neuron activity,inactivity, hypoactivity, and/or hyperactivity within an injury site orregion of tissue. The ultrasound images are correlated with the measuredneuroelectrical activity to identify one or more delivery zones for saidtherapeutic agent.

In another aspect of the disclosure, methods are provided to facilitatethe cellular implant therapeutic agents within an injury site of aspinal cord. Several parameters are recorded and analyzed to identify apreferred environment within an injury site for delivery of thetherapeutic agent. In preferred embodiments the environment is locatedwithin an acutely or chronically injured spinal cord, and the deliveredtherapy is the neuronal precursor cells or neurospheres of the inventionfor restoration or regeneration of tissue within the targeted site.

Preoperative Imaging

In various embodiments, injured sites of the CNS are identified byacquiring preoperative imaging data of the CNS. Various methods areknown in the art to obtain preoperative imaging data. Preoperativeimaging data includes Medical Resonance Imaging (MRI), computedtomography (“CT” or “CT scan”), x-ray data, and/or myelography.

MRI data are acquired by conventional procedures known in the art. Invarious embodiments, a strong magnetic field is applied to a patient'sbody. MRI is a non-invasive imaging method based on the use of radiowaves and a magnetic field to provide cross-sectional images of thespinal cord. (see for example Falci et al., Journal of Spinal CordMedicine, 22(3), 173-181 (1999)). The imaging information from thespinal cord is obtained and used to localize the site of the contusionor injury. In particular, the preoperative imaging data providesinformation regarding the size, shape and possible type of injury thatthe patient has suffered and facilitates the surgeons ability tolocalize the correct level of the spinal cord to expose. In addition,the preoperative imaging provides a view of the spinal cord's stability,the degree of spinal cord tethering, myelomalacic change, true cysticcavitation, and cerebrospinal fluid flow.

In some embodiments, the entire spinal cord can be imaged. In furtherembodiments, different slices of the spinal cord (e.g. the cervical,thoracic, or lumbar regions) can be imaged. Different depths of thespinal cord also can be imaged.

FIG. 1 depicts an exemplary MRI from a patient having a spinal cordinjury. The MRI depicts three basic zones within an acutely injuredspinal cord: a zone having reduced electrical activity, a zone havinghyperactive electrical activity and a zone having normal electricalactivity.

The zone having reduced electrical activity (electrophysiologicrecording) is shown in the top panel of the recording data. The area ofreduced activity corresponds to the cells within an injured spinal cordat a contusion site (reference letter A), i.e., the direct site wherethe injury occurred. The contusion site can be of various dimensions andis typically viewed on an ultra-sound as a dark space (corresponding toa signature of liquid and other similar density materials).

Intraoperative Ultrasound

Intraoperative ultrasound images of abnormal or injured regions of theCNS are acquired. In various embodiments, the preoperative images canthen be correlated to information gathered via intraoperative ultrasoundon the same spinal cord.

In various procedures, a laminectomy is performed on the injured spinalcord to expose the dura of the region of injury as determined from theimaging data. Intraoperative ultrasonography then provides a radiographof the region of injury and provides a detailed view of possible nervedamage, myelomalacia, healthy or normal tissue. For example,intraoperative ultrasound can be used to access the size of the injury,regions of myelomalacia, location of spinal cord tethering and cord androotlet motion. Cord tethering was recognized as a loss of normalsubarachnoid space, adherence of the cord and rootlets to the dura, andthe loss of the normal anterior-posterior motion of the spinal cordcorresponding to the patient's heartbeat and CSF pulsations.Myelomalacia can be recognized as regions of hyperechoicity. The regionand type of CNS (e.g. spinal locations) defects are thus preciselymapped out and identified.

FIG. 2 depicts an exemplary intraoperative ultrasound of a spinal cordcontusion. A contusion site can be seen in the ultra-sound as a darkspace (corresponding to a signature of liquid and other similar densitymaterials). Normal, uninjured tissue is defined by the lighter-coloredspace bordering the contusion area. The specific location and borderregion of injured tissue can thus be identified.

Measuring Neuroelectrical Activity

Using this information regarding the patient's injury site, theneuroelectrical activity of injured cells can then be determined withinzones of the injury site to identify active neuron activity. Specifictracts of tissue can be detected. In particular, correlating imagingdata and/or ultrasound data with the measured neuroelectrical activityof cells at the injury site is used to locate specific environments ofcellular activity. Based on this correlation, a therapeutic deliveryzone or zones within the injury site for delivery of therapeutic agentscan be identified, thus increasing the prospect of a positive therapyresult. The electrical signals can be used to identify sites foradministration of additional therapies during surgery, without requiringestimation of the location and border of the injury site that wouldotherwise require a separately acquired ultrasound.

In additional embodiments, detection of neuroelectrical activity can beused to identify the location of treatment sites independently ofadditional imaging methods. For example, the neuroelectrical activity ofthe spinal cord can be detected at and surrounding a region of damagedtissue in the spinal cord to identify zones of active neuron activity orinactivity neuronal activity. In further aspects, hyperactivity andhypoactivity can also be detected. Therapeutic compounds can then beadministered to the site identified by neuroelectrical activity.

In one embodiment, zones at an acute injury site can be identified. Asillustrated in FIG. 3, three basic zones within the acutely injuredspinal cord were identified: a zone having reduced electrical activity,a zone having hyperactive electrical activity and a zone having normalelectrical activity. The zone having reduced electrical activity(electrophysiologic recording) is shown in FIG. 3A. The area of reducedactivity corresponds to the cells within an injured spinal cord at acontusion site depicted in FIG. 3D at position A, i.e., directly at thecontusion site. Contusion sites can be of various dimensions and aretypically viewed on an ultra-sound as a dark space (corresponding to asignature of liquid and other similar density materials).

An area of cells showing normal or healthy neuroelectrical activity istypically caudal to a contusion site. A typical electrophysiologicrecording of spontaneous neuroelectrical activity for normal or healthycells from within the normal zone is shown in FIG. 3B. This regioncorresponds to healthy cells in the light-colored region of FIG. 3D(noted by reference letter B).

A third zone of cells adjacent to, or bordering, the acute injury siteshows a third type of electrical activity. This bordering zone of cellsaround the contusion site show hyperactive neuroelectrical activity, asis shown in FIG. 3C. The area of hyperactivity corresponds to the cellswithin the area of the ultrasound labeled with reference letter C ofFIG. 3D.

The third zone of hyperactive cells in acute injuries one year or less,more typically six months or less, and most typically in three months orless, is reduced to less than normal organized electrical activity overtime. This pattern suggests a substantial loss of neurons within thisbordering zone, until in chronic injury sites, no hyperactiveneuroelectrical activity is shown or is anticipated. The border zone ofcells represents a region which progressively undergoes cell death(apoptosis and necrosis) over a period of time (typically less than oneyear). As such, this region of cells represents a source of tissue at anacute injury location that is undergoing progressive neuronaldeterioration, and can be a favorable region for administeringtherapeutic agents.

In various embodiments, the border region of an acute injury siterepresents the zone of cells most likely to enhance the effects of theinjury, as these cells are shown to be the most likely to progressivelydie over time (within one year of injury). As such, this border zone ofcells represents a target site within the acutely injured cord forslowing cell death. Therapies intended to minimize cell death and losscan be delivered directly to the border zone of the acutely injuredcord, thereby concentrating the effectiveness of regenerative and/ornon-apoptotic drug approaches.

Different types of healthy tissues can be identified. For example,specific injuries within different types of neuronal tissue can also bedetected from electrophysiologic recordings of specific types and tractsof tissues. FIG. 4A shows an electrophysiologic recording of uninjuredtissue in the spinal thalamic tract. FIGS. 4B and 4C, respectively,depict electrophysiologic recordings of uninjured tissue in theposterior column lateral and medial, respectively. The posteriorcolumn-medial lemniscus pathway is the sensory pathway responsible fortransmitting fine touch and conscious proprioceptive information fromthe body to the cerebral cortex.

Changes in the electrical signals measured in specific tracts or columnsin a patient allow the specific location of an abnormality or injury tobe identified with high precision. FIG. 6 depicts an example of extrememyelomalacia and cystic necrosis in the tissue of a patient. Theabnormal tissue is identified by the absence of organized neuronalelectrical activity of low energy. Therapeutic agents can thus betargeted directly to a specific injury site either with or withoutultrasound imaging, and a specific tract within the injury zoneidentified by neuroelectrical measurements.

Alternatively, different regions of spinal cord tissue can bedistinguished by measurement of neurophysiological signals. For example,cellular tracts can be distinguished from dorsal root entry zones(DREZ). FIG. 5A depicts an electrophysiologic recording of thecorticospinal tract of a patient, and FIG. 5B depicts anelectrophysiologic recording of the dorsal grey matter of a patient.Each of FIGS. 5A and 5B show a high frequency signal corresponding tothe firing of multiple nerve cells. FIG. 5B however also includes anunderlying slow frequency signal corresponding to organized cellularfiring represented in the tract. The high frequency nerve signalcombined with the low frequency cellular signal identifies the tissue asa tract. Similarly, FIG. 7 also depicts a high frequency signalcorresponding to the fast firing of multiple nerve cells overlapping aslow frequency signal corresponding to organized cellular activity. Thehigh frequency nerve signal combined with the low frequency cellularsignal identifies the tissue as a tract in the column.

By contrast, FIG. 8 depicts the signal obtained from grey matter of adorsal root entry zone (DREZ). The high frequency corresponds to fastfiring of nerves at the DREZ. However, there is not underlying slowfrequency overlapping the high frequency signal. The DREZ signal is thusreadily distinguished from cellular tracts.

Therapeutic Agents

Therapeutic agents can be selected and targeted to specific types ofinjured tissue based on the correlation of imaging methods andneuroelectrical measurements.

Injury to the spinal cord often results in mechanical killing and damageto axons. Damage can include both grey and/or white matter within thespinal column. Typically, injuries to the cord include a second wave ofdamage, often caused by inflammation within the initially damaged area.The end result is a state of disrepair within the cord. Often axons arenot functional due to disconnection or loss of insulating myelin. Inother circumstances, glial scars form within the area of damage. Inother cases, axons remain intact and myelinated, but carry insufficientsignal volume to convey useful directives to the brain or muscles.

Recently, it has been determined that as little as 10% of the standardaxon complement would provide the capacity for an injured patient towalk, or that limiting an injury site to 0.5 inches×0.5 inches couldhave a significant impact on a patient's quality of life (McDonald,Repairing the Damaged Spinal Cord, 281(3):64-73). As such,administration of therapies of the spinal cord at targeted locationsprovide enhanced treatment of spinal cord injuries.

Various therapeutic agents can be delivered to a specific CNS locationusing the methods disclosed herein. For example, axon growth stimulatingfactors, inhibitor-neutralizing antibody or equivalent small molecules,netrins, corticosteroids, nerve grafts, extracellular matrix components,scaffolding components (e.g. Schwann cells), genetically engineeredfibroblasts, and various progenitor cells, for example mesenchymal stemcells, embryonic stem cells, etc. In various embodiments, combinationsof biologic factors and cell populations will be delivered together tomaximize the targeted environment and provide building blocks for repairwithin the injury site. Examples of these therapies include, but are notlimited to, those described in Tator et al., Neurosurgery 59(5) November2006. These include neuropharmaceuticals as well as cellular therapies.

Additional therapies can include embryonic stem cells, fetal olfactorybulbs, autologous olfactory mucosa, and stem cells described in Tator etal. (supra) can be used. Pharmaceuticals administered can includeminocycline, cethrin, and ATI355.

In various embodiments, stem cells can be administered. For example,bone marrow stem cells can be administered as whole stem cells. Bonemarrow cells can contain a mixture of hematopoietic cells, variousmononuclear cells, such as macrophages, and marrow stromal cells. Inother embodiments, peripheral blood stem cells can be administered. Infurther embodiments, human stem cells, including neuronal stem cells orpluripotent cells, human embryonic stem cells, or fetal Porcine StemCell Xenotransplants can be administered. Additional cellular therapiescan include human fetal spinal cord cells, olfactory ensheathing glia orolfactory Bulb cells can be administered.

In still other embodiments, a rho antagonist can be administered.Exemplary rho antagonists include kinase inhibitors (e.g., cethrin).Alternatively, an anti-Nogo-A inhibitor can be administered to the Nogoinhibitory protein associated with myelin. Nogo inhibitors can includeantibody or antibody-fragment based inhibitors.

In certain examples, cellular therapies can include isolation andexpansion of neural precursor cells in a manner that maintains andoptimizes their capacity to restore and/or regenerate function within aninjured CNS, and in particular within an injured spinal cord.Embodiments disclosed herein illustrate the utility of targeting thesecells into various types of CNS injury, and in particular, spinal cordinjury.

In certain embodiments, therapeutic agents can be administered toregions of damaged tissue to prevent expansion of initial damage andpromote secondary damage. For example, therapeutic agents that blockexcitoxic injury can be administered directly to the injured cells orborder region of cells. The administration of a therapy can beadministered, for example, to the hyperactive neuroelectrical signalobserved at the border region of an injury in FIG. 3C.

Alternatively, axonal regenerative therapy can be provided to thepatient. For example, therapeutic agents that overcome naturalinhibitors of regeneration can be provided to the axon at a specificlocation identified herein. Agents that induce axonal growth can also beadministered. For example, peripheral nerve bridges and neurotrophicfactors can be added to a cell.

In additional embodiments, compounds can be implanted into dead tissuecysts that can serve as scaffolds for axon regeneration.

In other embodiments, treatment may be delivered to demyelinated tissue.For example, chemicals can be delivered that prevent nerve impulses fromdissipating at demyelinated areas. Further, agents can be provided thatspur surviving oligodendrocytes to remyelinate axons. For example,exogenous myelin-forming cells can be transplanted.

In further embodiments, undiffentiated stem cells or precursor cells canbe administered directly to an injury location identified by thetechniques described herein. For example, neurospheres can beadministered to restore or regenerate damaged CNS tissue. In general,these cells are shown to have potent regenerative capacity in thetreatment of various CNS injuries. Embodiments of the invention providemethods for procurement of the precursor cells to the neurospheres.Target tissue having neuronal precursor cells is harvested and dissectedin accordance with approved techniques. For example, embryonic/fetaltissue is identified and dissected under sterile conditions. Thegestational age of the tissue, including size and anatomical landmarks,is used to identify areas of expected neuronal precursor cells. Theprocedure can be accomplished under a dissection microscope. In general,pieces of the forebrain and spinal cord are dissected from theembryonic/fetal tissue, freed of meninges and visible blood vessels, andrinsed in serum free culture medium. Note that preferred embodimentsutilize human embryonic/fetal tissue sources.

Various combinations of therapies are discussed in McDonald, supra,which is herein incorporated by reference in its entirety.

Screening Therapeutic Agents

In various embodiments, injured cells can be screened to determine theeffectiveness of a given therapeutic agent on a specific tract of cells.

In one example, effective treatments can be screened or tested toidentify specific treatment targets for cells at the injury site, borderzone, and normal zone depicted in FIGS. 3A-D. A sample of both thehyperactive, neuronal degenerating cells in the border zone and normalcells in the normal zone can be taken from a patient. The sample can betaken by any method known in the art. In certain embodiments, samplescan be taken by biopsy from the patient, for example a 1 mm×1 mm×2 mmsection of target tissue in the target zone. Note also that each sampleis isolated and treated separately—to minimize any potential forcross-contamination between samples. The samples removed from thepatient are analyzed using one or more biochemical/molecular techniquesto identify effectors present or at higher concentrations in the borderzone than in the normal zone sample. These techniques, for exampledifferential gene expression, protein separation by electrophoresis,etc., can be found in any of several well-known references, such as:Molecular Cloning: A Laboratory Manual (Sambrook et al. (1989)); GeneExpression Technology (Methods in Enzymology, Vol. 185, edited byGoeddel (1991) Academic Press, San Diego, Calif.); “Guide to ProteinPurification” in Methods in Enzymology (Deutshcer, 3d., (1990) AcademicPress, Inc.), PCR Protocols: A Guide to Methods and Applications (Inniset al. (1990) Academic Press, San Diego, Calif.); and Gene Transfer andExpression Protocols, pp 109-128, ed. Murray, The Humana Press Inc.,Clifton, N.J.). Note also that the analysis may alternatively providedata that the hyperactive border zone cells are devoid of neuronaldegenerating effectors or express a potential inhibitor of neuronaldegeneration at a lower concentration than in the normal tissue. In anyevent, the methods will provide the identification of neuronaldegeneration.

In alternative embodiments, injured and normal specific spinal tractscan be screened for targets or cellular markers. For example, samples ofinjured and normal white matter or dorsal grey matter can be biopsiedand tested against various treatments prior to providing the treatmentto the patient. Alternatively, injured or normal tissue of differenttracts can be tested to identify markers or effectors specific to aninjury location. Samples removed from the patient can be analyzed usingone or more biochemical/molecular techniques to identify effectorspresent or at higher concentrations in various tracts and injury zones.

In alternative embodiments, a therapeutic agent can be screened foreffectiveness on different spinal tracts. For example, samples ofinjured and normal white matter or dorsal grey matter can be biopsiedand tested against various treatments prior to providing the treatmentto the patient. The effectiveness of a given therapy on a specific celltype can be determined by in vitro assays.

The disclosure will be more readily understood by reference to thefollowing example, which is provided by way of illustration and is notintended as limiting.

EXAMPLE

MRI, Ultrasound and Electrical Signature Map Target Sites For Deliveryof A Cellular Therapy in an Injured Spinal Cord

Clinical Materials and Methods:

A combination of data from one or more patients has been used toillustrate one embodiment of the present invention. Data was selected toillustrate one or more aspects for targeting and delivery of a cellularor biologic therapy to an injury site. Data for the present inventionwas obtained from patients who have sustained traumatic thoracolumbarspinal cord injuries.

Patients eligible for cellular or biological therapy to the spinal cordtypically have had an injury causing significant damage to one or moreregions of the spinal cord.

Preoperative Procedures:

A patient scheduled to undergo the methods of the present invention willundergo preoperative evaluation with one or more of: plain x-ray,magnetic resonance imaging (MRI), and/or cat scan (CT) myelography. Thecombination provides an evaluation of the spinal cord injury site. Priorto surgical treatment in accordance with the present invention, apatient would undergo pharmacologic treatment appropriate for spinalcord injury such as methylprednisolone in the acute phase, and any othermedications needed to treat spasticity, autonomic dysreflexia,neuropathic pain, or any other symptoms related to the spinal cordinjury including administration of oral tricyclic antidepressants (TCA),antiseizure medication, Baclofen, Klonopin, and narcotic analgesics. Insome instances, a pump can be placed for intrathacal infusion ofnarcotics, Baclofen, Clonidine or local anesthetic. FIG. 1 illustratesan MRI from a patient having an injury to the spinal cord consistentwith a contusion and regions of cystic necrosis, myelomalacia andregions of healthy cord. The MRI gives an initial indication as to thedimensions of the injury site, time frame from which the injuryoccurred, potential boundaries within which a cellular or biologictherapy would be delivered.

Intraoperative Ultrasound and DREZ Recording

Multilevel laminectomies were performed on the patient as is known toone of skill in the art. Briefly, laminectomies were performed to exposethe spinal cord at the level of injury, as well at uninjured levelscephalad and caudal. Spinal levels for potential delivery of a cellularor biologic therapy were determined by the preoperative proceduresdescribed above. An intraoperative ultrasonography was performed tobetter identify the injury site.

Measuring Neuroelectrical Activity

The neuroelectrical activity was measured to determine the electricalsignal at different locations of the acute injury. An active electrodewas inserted into different locations of the DREZ tissue. The activeelectrode used was a 25 mm TECA MF 25 monopolar electrode with thedistal 2 mm exposed. The electrode was implanted at each tissue locationwith use of the intraoperative microscope to a 2 mm depth. Ground andreference Glass subdermal (EEG) electrodes were placed in exposedparaspinous muscle bilaterally. Spontaneous electrophysiologicrecordings were made with a Cadwell Spectrum 32 evoked potentialaverager at a gain setting of 50 with the high frequency filter set at 3KHz and the low frequency filter at 100 Hz. The recordings were onesecond in duration. Additional evaluation with fast fourier transform,root mean square analysis, and spindle analysis were made.

1. A method of delivering a therapeutic agent to the central nervoussystem (CNS) of a patient comprising: obtaining preoperative imagingdata of the CNS of the patient to determine boundaries of a region ofdamaged tissue in said the CNS of the patient; obtaining one or moreintraoperative images of said region of damaged tissue; measuringneuroelectrical activity in and surrounding the region of damaged tissuein one or more tracts of grey and white matter to identify zones ofactive neuron activity, inactivity, hypoactivity, and/or hyperactivity;correlating said ultrasound images with the measured neuroelectricalactivity at and surrounding the region of damaged tissue to identify oneor more delivery zones for said therapeutic agent; delivering thetherapeutic agent to the one or more delivery zones.
 2. A method ofdelivering a therapeutic agent to the central nervous system (CNS) of apatient comprising: measuring neuroelectrical activity in andsurrounding the region of damaged tissue in the spinal cord of saidpatient to identify zones of active neuron activity, inactivity,hypoactivity, and/or hyperactivity to identify one or more deliveryzones for said therapeutic agent; delivering the therapeutic agent tothe one or more delivery zones.
 3. The method of claim 1, wherein thepreoperative image data is selected from the group consisting of a MRI,a CT scan, and an X-ray scan.
 4. The method of claim 1, wherein thepreoperative image data is a MRI.
 5. The method of claim 1, wherein saidone or more tracts are selected from the group consisting of the spinalthalamic tract, the posterior column, the corticospinal tract, anddorsal grey matter.
 6. The method of claim 1 or 2, wherein said deliveryzone comprises a regions of hyperactivity.
 7. The method of claim 1 or 2wherein the therapeutic agent promotes axonal regeneration.
 8. Themethod of claim 1 or 2, wherein the therapeutic agent promotesremyelination.
 9. The method of claim 1 or 2, wherein the therapeuticagent inhibits nerve impulses from dissipating in demyelinated areas.10. The method of claim 1 or 2, wherein the therapeutic agent inhibitsexotoxic shock.
 11. The method of claim 1 or 2, wherein the therapeuticagent comprises stem cells.